Development and characterization of candidate rotavirus vaccine strains derived from children with diarrhoea in vietnam

Vaccine 27S (2009) F130–F138 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Development and characterization of candidate rotavirus vaccine strains derived from children with diarrhoea in Vietnam Luan T Le a,∗ , Trang V Nguyen b , Phuong M Nguyen b , Nguyen T Huong a , Ngo T Huong a , Nguyen T.M Huong a , Tran B Hanh a , Dang N Ha a , Dang D Anh b , Jon R Gentsch c , Yuhuan Wang c , Mathew D Esona c , Roger I Glass d , A Duncan Steele e , Paul E Kilgore f , Nguyen V Man a , Baoming Jiang c,∗∗ , Nguyen D Hien a a Center for Research and Production of Vaccines and Biologicals (POLYVAC), 135 Loduc Street, Hai Ba Trung, Hanoi, Viet Nam National Institute of Hygiene and Epidemiology, Hanoi, Viet Nam c Division of Viral Diseases, U.S Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA d Fogarty International Center, National Institutes of Health, Bethesda, MD, USA e Vaccines and Immunization, PATH, Seattle, WA, USA f International Vaccine Institute, Seoul, Republic of Korea b a r t i c l e Keywords: Rotavirus vaccine Diarrhoea Vietnam i n f o a b s t r a c t In Vietnam, rotavirus infection accounts for more than one-half of all hospitalizations for diarrhoea among children less than years of age While new vaccines to prevent rotavirus diarrhoea have been developed and introduced into some countries by multinational manufacturers, the ability for developing countries such as Vietnam to introduce several new and important vaccines into the routine infant immunization schedule may be challenging In order to be partially self-sufficient in vaccine production, Vietnam has pursued the development of several rotavirus strains as candidate vaccines using isolates obtained from Vietnamese children with diarrhoea This paper describes the origin, isolation and characterization of human rotavirus strains being considered for further vaccine development in Vietnam The goal is to prepare a monovalent G1P [8] rotavirus vaccine using one of these strains obtained in Vietnam and naturally attenuated by multiple passages in cell culture While this is an ambitious project that will require several years’ work, we are using the lessons learned to improve the overall quality of vaccine production including the use of Vero cell techniques for the manufacture of other vaccines in Vietnam © 2009 Elsevier Ltd All rights reserved Introduction From 1998 to 2003, we conducted sentinel hospital surveillance of rotavirus diarrhoea among children less than years of age seen in four cities of Vietnam Rotavirus was identified in more than 50% of children hospitalized for diarrhoea [1,2], indicating a strong need for a vaccine that could substantially reduce Abbreviations: US CDC, United States Centers for Disease Control and Prevention; EIA, enzyme immunoassay; GCP, good clinical practice; GLP, good laboratory practice; GMP, good manufacturing practice; MS, master seed; PMKC, primary monkey kidney cells; PRKC, primary rabbit kidney cells; MA104, African green monkey kidney cells; PAGE, polyacrylamide gel electrophoresis; PL, passage level; POLYVAC, Center for Research and Production of Vaccines and Biologicals, Hanoi, Vietnam; RT-PCR, reverse transcription-polymerase chain reaction; WHO, World Health Organisation; WS, working seed ∗ Corresponding author Tel.: +84 48213820; fax: +84 48213203 ∗∗ Corresponding author Tel.: +1 4046392861; fax +1 4046393645 E-mail addresses: luanpolyvac@gmail.com (L.T Le), bxj4@cdc.gov (B Jiang) 0264-410X/$ – see front matter © 2009 Elsevier Ltd All rights reserved doi:10.1016/j.vaccine.2009.08.086 severe rotavirus hospital admissions, outpatient visits, and associated deaths In an analysis of rotavirus strains identified from prospective surveillance, G1P[8] was the most commonly identified strain (45%) The other common rotavirus strains—G2P[4], G4P[8], G4P[6] and G9P[8], were also present, and a few rare strains were found (e.g., G1P[4] and G9P[4]) Vietnam has a strong history of self sufficiency in local manufacturing of vaccines While many new vaccines are becoming available for immunization of children, the government is concerned about the affordability of these products and has encouraged the domestic manufacture of many new vaccines In recent years, the government, with international partners, has developed new manufacturing capacity for vaccines against Hepatitis B, Japanese B encephalitis, and measles [3,4], and based on the high burden of rotavirus disease, has recently provided seed funding to develop rotavirus vaccine The demonstration that a live, oral rotavirus vaccine could be produced from a single human strain, attenuated by multiple passages, and manufactured in Vero cells, encouraged us to apply a L.T Le et al / Vaccine 27S (2009) F130–F138 F131 Table Adaptation and passage history of the candidate human rotavirus vaccines for use in Vietnam Strain Origin KH 0118 6-month-old girl in Khanh Hoa Hospital, Nha Trang, 2003 SP 210-2000 11-month-old boy in Saint Paul Hospital, Hanoi, 2000 SP 203-2001 9-month-old boy in Saint Paul Hospital, Hanoi, 2001 Genotype G1P[8] G1P[4] G4P[6] Passage history Stool ↓ Isolation in MA104 cells (3×) ↓ Plaque purification in MA104 cells (1×) ↓ Passages in MA104 cells ↓ 12 Passages in PMKC* ↓ 18 Passages in Vero cells ↓ Cloning by limiting dilution times in Vero cells ↓ Passages in Vero ↓ Master virus seed (PL5)* ↓ Working virus seed (PL6) Stool ↓ Isolation in MA104 cells (3×) ↓ Plaque purification (3×) in MA104 cells ↓ Passages in MA104 cells ↓ 11 Passages in PMKC ↓ 10 Passages in Vero cells ↓ Cloning by limiting dilution times in Vero cells ↓ Passages in Vero ↓ Master virus seed (PL5) ↓ Working virus seed (PL6) Stool ↓ Isolation in MA104 cells (3×) ↓ Plaque purification (2×) in MA104 cells ↓ Passages in MA104 cells ↓ 24 Passages in PMKC ↓ 12 Passages in Vero cells ↓ Cloning by limiting dilution times in Vero cells ↓ Passages in Vero ↓ Master virus seed (PL5) ↓ Working virus seed (PL6) Note: * PMKC: primary monkey kidney cells; PL: passage level similar strategy in Vietnam since these methods are already used in our existing vaccine production activities [5] In addition, in the process of developing a rotavirus vaccine, we have been able to update some methods and procedures used in the manufacturing of other vaccines Finally, by adapting a local rotavirus strain with several additional strains as backup candidate rotavirus vaccine strains, we will be better able to match vaccine viruses to local rotavirus strains in circulation and thus, achieve an equivalent or higher level of protection compared with currently available rotavirus vaccines Materials and methods 2.1 Overall strategy Our strategy to develop a live, oral rotavirus vaccine for Vietnam has involved the selection of the most common circulating serotype, G1P[8] recovered from an 6-month-old child with rotavirus diarrhoea captured in the national surveillance program along with two other strains (G1P[4] and G4P[6]) detected in young infants These strains were adapted and passaged in cell culture to select the isolates that grew best and to highest titre in Vero cells The strains were also fully characterized, and proceeded through at least 40 blind passages with the assumption that natural attenuation would occur over time Following testing of the candidate vaccine strains which included toxicity testing and animal studies, our next steps include human studies: to ensure the safety of the strains, to determine if they have been adequately attenuated for infants, and to ensure that they still induce a robust immune-response This paper describes the selection, passaging, initial characterization and preliminary safety testing of our selected strains For guidelines, we followed the WHO published procedures and methods to develop oral rotavirus vaccines [6] All work was conducted in a special laboratory at the Centers for Disease Control and Prevention (CDC) in Atlanta and POLYVAC, a facility approved by national authorities in Vietnam for the production of vaccines for polio and measles 2.2 Origin and properties of Vietnam human rotavirus vaccine candidates Three faecal specimens from otherwise healthy Vietnamese infants with acute diarrhoea, that tested positive for rotavirus by EIA and PCR and contained representative genotypes G1P[8], G1P[4], and G4P[6] of rotavirus, were selected for further development (Table 1) [7,8] These strains were first adapted to grow in MA104 cells, a cell line derived from monkey kidney cells To avoid concerns about contamination with the agent of bovine spongiform encephalopathy, we used MA104 cells that were originally received in January 1979 at passage 14 from MA Bioproducts which has changed to Bio Whittaker (Walkersville, MD) and had been stored at the Cell Culture Laboratory, Biologics Branch, US CDC We have complete passage history and full documentation for these MA104 cells Rotavirus strains were purified and serially passaged in MA104 cells and primary monkey kidney cells (PMKC) to increase the rotavirus virus titres A final stage included passage in Vero cells qualified for vaccine production of a master and working virus seed The PMKC were drawn from the production facility for OPV which is operated by POLYVAC and has previously undergone rigorous routine procedures for safety testing as required for OPV vaccine production under Vietnamese national regulatory procedures The human rotavirus (HRV) strain G1P[8] (KH0118) was obtained from a 6-month-old girl in Khanh Hoa General Hospital (Nha Trang, Vietnam) in 2003 This is the most commonly occurring strain globally and also in Vietnam and proof of principle exists with the monovalent Rotarix® vaccine [5] This virus was isolated by passaging times and plaque-purified once in MA104 cells It was further passaged additional times in MA104 cells and then 12 additional times in PMKC The virus was then adapted and passaged 18 times in Vero cells that were qualified for use in vaccine production Individual clones were selected by limiting dilution twice in Vero cells After four more passages in Vero cells, a master seed (MS) was produced at passage level (PL) One additional passage of MS in Vero cells produced a working seed (WS) of PL for a total passage number of 48 The HRV strain G1P[4] (SP210-2000) was derived from an 11month-old boy in Saint Paul Hospital (Hanoi, Vietnam) in 2000 The virus was isolated, plaque-purified times and passaged more times in MA104 cells (Table 1) The virus was then passaged 11 times in PMKC and adapted to grow in Vero cells (10 passages) After cloning by rounds of limiting dilution, the virus was amplified in Vero cells to prepare MS (PL5) and WS (PL6) for a total passage number of 44 F132 L.T Le et al / Vaccine 27S (2009) F130–F138 A G4P[6] strain (SP203-2001) was identified from a 9-monthold boy in Saint Paul Hospital in 2001 The virus was isolated by initial passage and plaque purification twice, and passaged times all in MA104 cells After 24 passages in PMKC, the virus was adapted to grow in Vero cells (12 passages) After rounds of purification by limiting dilution, the virus was amplified in Vero cells to prepare MS (PL5) and WS (PL6) for a total passage number of 56 2.3 Quality control of master and working rotavirus seeds The MS and WS for each virus were tested for adventitious agents by using an array of in vitro and in vivo assays as suggested in the WHO guidelines for the production of an attenuated rotavirus vaccine [6] The tests used were approved by the national regulatory authority in Vietnam The MS and WS were routinely checked for identity by 10% PAGE (Fig 1) and by RT-PCR (G and P typing) to assure the correct electropherotypes and G and P genotypes, respectively using published methods [7,8] Additional tests were performed on the virus seeds to confirm the stability of the viral genome and absence of adventitious agents over a defined passage range The passage level used to qualify the virus seeds is generally 5–10 passages beyond that used for vaccine production [6] Our working virus seed (PL6) is just one more passage of the master virus seed (PL5) in Vero cells 2.4 Safety tests Each master or working seed lot was tested for toxicity, safety and immunogenicity in animals including guinea pigs, mice, rabbits, and monkeys (Table 2) They were also tested by RT-PCR for adventitious viruses, including foamy virus, hepatitis viruses and mycoplasma, by the Research and Development Department at the Company for Vaccine and Biological Production No1 in Hanoi [9–11] Safety tests for adventitious viruses were also conducted in cell cultures including PMKC, neonatal rabbit kidney cells, Vero and Hep cell lines as recommended by the WHO Guidelines [6] 2.5 Analysis of NSP4, VP6, VP7 and VP4 genes of candidate vaccines The MS of the candidate vaccines was subjected to sequence analysis for rotavirus genes 4, 6, and 10, which encode the two neutralization antigens, the group antigen and the putative viral enterotoxin Partial nucleotide sequences of genes and (encoding VP4 and VP7, respectively) were obtained from amplicons generated in the first round of G and P typing RT-PCR Phylogenetic analysis was performed on the deduced amino acid sequences from aa 1-175 for NSP4, 1-397 for VP6, 1-288 for VP7 and Fig RNA profiles of rotavirus vaccine candidates, G1P[8], G1P[4] and G4P[6] isolated in Vietnam Rotavirus dsRNA was extracted from infected Vero cell cultures and analyzed on 10% polyacrylamide gel, followed by staining with silver nitrate Lanes 1, DNA marker; 2, G1P[8] vaccine seed; 3, G1P[4] vaccine seed; 4, G4P[6] vaccine seed, and 5, reference Wa strain 16-278 for VP4 Sequence data were first analyzed using CHROMAS software (Technelysium Pty Ltd, Tewantin, Australia) Forward and reverse sequence data of each sample were aligned using the Edit Seq and MegAlign program (DNASTAR Inc., Software, Madison, WI) to obtain the final consensus sequence Complete alignment was performed with Cluster W (DNASTAR Inc., Software, Madison, WI) Table Qualification tests used for master and working rotavirus seeds and results * Qualification tests Methods Results Hepatitis A virus Hepatitis B virus Hepatitis C virus Simian virus SV40 Simian foamy virus Mycoplasma Bacteria and fungi M tuberculosis Tissue culture safety: Vero, Hep2, PMKC, PRKC* Animal safety: adult mice, rabbits, Guinea pigs Safety and immune-response in young monkeys (6–12-month-old) PCR PCR PCR PCR PCR PCR Bacterial and fungal culture Mycobacterial culture Cytopathic effect, hemedsorption Observation Observation Negative Negative Negative Negative Negative Negative Negative Negative Negative Increase in weight and appear healthy Increase in weight and appear healthy Measurement of neutralizing antibody by plaque reduction assays 3-fold increase in serum antibody titres from pre- to post-third dose vaccination Note: PCR: polymerase chain reaction; PMKC: primary monkey kidney cells; PRKC: primary rabbit kidney cells L.T Le et al / Vaccine 27S (2009) F130–F138 F133 Fig Phylogenetic tree of the NSP4 gene fragments of the Vietnam candidate vaccines The Vietnamese strains used to develop vaccine candidates are indicated in the boxes The 738 bp PCR product of NSP4 gene was amplified using Jrg30 and Jrg31 primer pair and directly sequenced Abbreviations of countries of isolation in strain names: AU (Australia), BL (Bangladesh), CN (China), ID (India), JP (Japan), TL (Thailand), TW (Taiwan) and US (the United States of America) Results 3.1 Preparation of candidate rotavirus vaccines Three rotavirus strains (rotavirus genotypes G1P[8], G1P[4] and G4P[6]) from patients enrolled in the surveillance study were selected as candidate strains for rotavirus vaccine development All patients had diarrhoea with different degrees of dehydration but no apparent fever The patients were otherwise in good health We first adapted rotavirus strains to grow in MA104 cells that were prepared before 1980 with complete detailed documentation and then passaged the strains in PMKC and qualified Vero cells using certified reagents under stringent GLP conditions (Table 1) Strains were purified by plaque assay and limiting dilution MS and WS banks were prepared with Vero cells in roller bottles 3.2 Preclinical safety and immunogenicity of candidate rotavirus vaccines The MS and WS of the candidate rotavirus vaccine strains were shown to be free of extraneous agents (hepatitis A, B and C viruses, SV40, foamy virus, mycoplasma, bacteria and fungi) and also appeared to be safe in tissue culture, laboratory animals including monkeys (Table 2) All vaccines elicited a 3-fold increase in serum F134 L.T Le et al / Vaccine 27S (2009) F130–F138 Fig Phylogenetic tree of the VP6 gene fragments of the Vietnam candidate vaccines The Vietnamese vaccine candidates are indicated in the boxes The full length of VP6 gene (1356 bp) was amplified using Jrg7 and Jrg8 primer pair and directly sequenced using PCR and internal primers Abbreviation of countries of isolation in strain name: AU (Australia), BL (Bangladesh), CN (China), JP (Japan), ID (India), NZ (New Zealand), PLP (Philippines), TL (Thailand) and US (The United States of America) neutralizing antibody titres following immunization with vaccine doses compared to pre-vaccination titres in baby monkeys (data not shown) All strains had long RNA electropherotypes (Fig 1) Of note, the profile of the G1P[4] strain demonstrated a high level of similarity to that of reference G1P[8] strain Wa The G and P types of these candidate vaccines were confirmed by RT-PCR and sequence analysis 3.3 Strain characterization—sequencing 3.3.1 NSP4 typing The NSP4 sequences of candidate rotavirus vaccine strains were compared with 175 aa sequence fragments of viruses isolated from Bangladesh, India, Thailand and China in the same period and with reference strains Wa, DS-1, ST-3 and M37 NSP4 gene of the KH0118 (G1P[8]) and SP203-2001 (G4P[6]) strains belong to genogroup B (prototypes Wa, ST-3 and M37), whereas the SP210-2000 (G1P[4]) strain belongs to genogroup A (prototype DS-1) (Fig 2) We observed several notable differences in the NSP4 sequence between the Vietnamese strains in this study, the reference G1P[8] strain, and strains from other countries In particular, there were substitutions of Thr to Met at aa45, Ile to Val at aa76, Tyr to Cys at aa85, and Ser/Lys for Iso at aa97 (data not shown) The position aa131 in the NSP4 toxic peptide (aa114-135) was Tyr for G1P[4] and His for G4P[6] and G1P[8] strains, consistent with the ability of the original strains of these vaccine candidates to cause diarrhoea in children However, all of these strains have the Ile at aa 135, a marker for attenuation [12], indicating that the strain might have been attenuated after 40 or more passages in cell culture The single strain from Vietnam (KH210-2004) whose NSP4 sequence was previously available also showed high aa sequence similarity to our G1P[8] strain and came from the same province in Vietnam 3.3.2 VP6 typing The Vietnamese vaccine candidates belong to two major VP6 genogroups The region of aa280-350 defines VP6 genogroups [13] L.T Le et al / Vaccine 27S (2009) F130–F138 F135 Fig Phylogenetic tree of the VP7 gene fragments of the Vietnam G1P[8] and G1P[4] (4a) and G4P[6] (4b) candidate vaccines Vietnamese vaccine candidates are indicated in the box The 896 bp PCR products were amplified using 9con1L and VP7R primers and directly sequenced using PCR and internal primers Abbreviations of countries of isolation in strain names: Au (Australia), BL (Bangladesh), BRZ (Brazil), CN (China), ID (India), ISR (Israel), JP (Japan), TL (Thailand), TW (Taiwan), US (The United States of America) and VN (Vietnam) and residues RPPN at aa 296-299 are present in all Vietnam strains, suggesting that they belong in subgroup I or II (Fig 3) The G1P[8] and G4P[6] strains showed characteristics of genogroup II (Asn at aa 305, Asn at aa 339, Leu at aa 342 and Ala at aa 348), while the G1P[4] showed characteristics of genogroup I and therefore of subgroup I (Ala at aa 305, Asn at aa 310, Glu at aa 315, Ser at aa 339, Met at aa 342 and Ser at aa 348) Phylogenetically, the Vietnam G1P[4] was in the same cluster as DS-1, SA11, 1076 and AU-1, whereas the Vietnam G1P[8] and G4P[6] strains were in the same cluster with Wa, RV3 and ST-3, confirming the classification of our vaccine strains (Fig 3) The sequence of VP6 from our G1P8 strain was similar to those from Bangladesh/2002–2003 and Thailand/2001 whereas the sequence of G1P[4] strain was closely related to those in Bangladesh/2000 and India during the same period and to DS-1 and SA11 (Fig 3) In addition, the Ala at aa172 of the Vietnam G1P[4], DS-1 and SA11 (subgroup I) was distinctly different from the Met at this position for many other subgroup II strains including the Vietnam G1P[8], G4P[6], Wa, RV3 and ST-3 F136 L.T Le et al / Vaccine 27S (2009) F130–F138 Fig Phylogenetic tree of the VP4 gene fragments of the Vietnam candidate vaccines The Vietnamese vaccine candidates are indicated in the box The 876 bp PCR product was generated using primer pair con3 and con2 and directly sequenced using PCR and internal primers Abbreviations of countries of isolation in strain names: Au (Australia), BL (Bangladesh), CN (China), HGR (Hungary), ID (India), JP (Japan), Korea (KOR), TL (Thailand), UK (United Kingdom), US (The United States of America), VZL (Venezuela) and VN (Vietnam) The Vietnam G1P[4] strain also showed amino acid substitutions of unknown importance at positions aa330 (Val-Ile), aa369 (Glu-Asp) and aa396 (Ile-Val) compared with other strains 3.3.3 VP7 sequences Vietnam during the same period (2003), and similar to those from Bangladesh and Thailand, clustered into lineage III, but different from those isolated from Japan and China or Finland (other lineages I, II and IV) (Fig 4a) Within the same lineage III, the Vietnam G1P[4] and G1P[8] were quite different The VP7 sequences of the Vietnam G1 candidate vaccine strains were similar to other Vietnamese strains (G1-lineage III) whereas the VP7 sequence of the G4 strain was similar to the corresponding gene of other strains from Vietnam, China, Japan, and Thailand and the porcine Gottfield strain (Fig 4) The VP7 sequence fragments of the two G1 strains were similar to those isolated elsewhere in 3.3.4 VP4 sequence The amino acid sequence of VP4 from the Vietnamese G1P[8] vaccine candidate strain demonstrated that strain KH0118 (G1P[8]) falls in the P[8]-2 F45-like lineage, having a high degree of homology with OP354 strain P[8]-3 (Fig 5) Many of the Vietnamese L.T Le et al / Vaccine 27S (2009) F130–F138 G1P[8], G4P[8] and G9P[8] strains were in the P[8]-2 lineage, and some G3P[8] and G9P[8] are of P[8]-3 lineage [14] Due to differences in the region sequenced between the VP4 sequences in our study and the previous study, these strains could not be included in the phylogenetic tree Amino acid alignments within the same region revealed the conserved Pro at aa224 and 225, like many other P[8] rotavirus strains Important substitutions in VP4 sequence of the Vietnam G1P[8] included Arg-Thr (aa144) and Arg-Gly (aa268) (data not shown) The VP4 sequence of the P[4] strain was similar to other Vietnamese G2P[4] strains isolated elsewhere The G1P[4] strain had important substitutions in the VP4 sequence compared to other strains including changes of Thr-Ala at aa103 and Arg-Gly at aa162 The Vietnam G1P[4] strain shared 96.5–98.1% homology with TBchen 2004 and Kmr029-2001 strains in the same cluster, and 92.6% and 95.3% homology with DS-1 and RV5 of different clusters, respectively (Fig 5) By contrast, the VP4 sequence of the G4P[6] strain shows high similarity to porcine strains The VP4 sequences of P6 strains from Vietnam are alike and clustered into a separate lineage with those reference strains isolated in 1980s (RV3, ST-3 and M37) and strains from Taiwan and Bangladesh The G4P6 clusters in the same lineage as the VN904 (G9P[6]), VN846 and VN592 (both are G4P[6] strains) This lineage was identified as P6-Ic, which has more than 95% similarity to porcine strains 221/04 and 134/04 [14] The Vietnam G4P[6] did not have the same conserved residues as other P6 strains at aa 90 and 91, Pro-Ile instead of Qln-Val, aa98 Arg instead of Lys, aa101 Val instead of Ile, aa140 Tyr instead of Phe, aa170-172 Gly-Gly-Arg instead of Tyr-Asn-Ser, aa202 Thr instead of Val, aa232 Val instead of Ile, aa242 Ala instead of Val, and aa255 Val instead of Ile The Vietnam G4P[6] strain exhibited many substitutions at the VP4 gene compared to other reference and field strains in the Asian region Thus, our G4P[6] strain might have porcine origins Discussion This is the first report to describe the development of candidate rotavirus vaccines from human strains G1P[8], G1P[4] and G4P[6] isolated in Vietnam These strains were adapted for growth, cloned, and prepared as MS and WS banks in Vero cells The G1P[8] strain could potentially be used as a monovalent vaccine with a much lower production cost and a viral sequence that fits better with the circulating P[8] lineage in Vietnam The other two strains (G1P[4] and G4P[6]) may be included in a vaccine formulation, if needed, to improve cross-protection and enhance overall vaccine efficacy Further studies will be needed to determine whether the G1P[4] vaccine strain induces immunologic cross-protection against both G1 and G2 viruses, derived from different rotavirus genogroups Additional evaluation of the candidate rotavirus vaccine strains is needed to assess whether or not the multiple passages in cell culture led to attenuation of the virus strain, something that can now only be determined in human studies We have characterized the HRV strains that have been used for the development of candidate rotavirus MS G1P[8], G1P[4] and G4P[6] These strains represented the major P and G genotypes (G1) that have been circulating in Vietnam [2] Results of surveillance in Vietnam indicate that a P[8] strain can cover over 70% of the serotypes in circulation [2] We also developed P[4] and P[6] vaccine candidates to cover other major P types (14.4% and 7.6%, respectively) of rotavirus strains circulating in Vietnam The virus seeds were biologically cloned by limiting dilution in Vero cells to enhance genetic homogeneity as well as to remove potential adventitious agents that may have been present in the initial virus inocula or derived from the attenuation process [5,6] The virus seeds were examined by performing a variety of tests and were qualified to be F137 free of adventitious agents [6] The vaccine candidates were also demonstrated to be free of adventitious agents in cell culture and in experimental animal models All vaccine candidates induced neutralizing antibody response in young monkeys Additional sequence analysis of the NSP4 and VP6 genes of the strains indicates that our vaccine candidates covered the major NSP4 genotypes and the subgroups of VP6 Sequence of the NSP4 toxic peptide identified one of the markers for attenuation (Ile at aa135), probably acquired during repeated passages in the cells, yet still showed Tyr/His at aa131, characteristic of the original strains causing diarrhoea [12] The VP7 and VP4 sequences of the G1P[8] strain shared similarity to some strains from Vietnam, Bangladesh, Thailand, whose sequences are available for analysis [14] Analysis of the VP4 sequence indicates that the Vietnamese P8 strain belongs to cluster F45-like (P[8]-2), whereas Rotarix® , one of the currently licensed and available rotavirus vaccines, is P[8]-1 [14] The findings that most strains in Vietnam fall into P[8]-2 and P[8]-3 suggest that it may be important to develop a rotavirus vaccine from local strains Several lines of rationale have led us to consider developing a new vaccine for Vietnam First, the long-term costs associated with introduction of several new vaccines may be unsustainable for a developing country such as Vietnam and the tradition of being selfsufficient in vaccine production has led the government to invest heavily in vaccine production and to support this development activity A single strain of rotavirus prepared as a vaccine through multiple passaging and preparation in Vero cells requires a level of technology similar to the production of OPV This production technology has been developed at institutions within Vietnam and has been adapted for use in development of the rotavirus vaccine candidates reported here Of note, the UK vaccine prepared from bovine reassortant strains has been licensed by US NIH to emerging manufacturers in eight countries and could be a direct and less expensive competitor vaccine to the Merck vaccine, RotaTeq® This vaccine is much more complicated to prepare as it contains multiple strains that need to be formulated together The epidemiology of rotavirus in Vietnam is more similar to the epidemiology in a middle income country rather than a poor developing country Therefore, we hope that our candidate G1 strains might perform as well in Vietnam as the monovalent GSK vaccine performs in Latin America and South Africa [5] Our vaccine strains have a sequence more homologous to other strains in Vietnam than strains elsewhere, including the G1P[8] strain in the GSK vaccine Therefore, these strains might well provide a slightly better level of protection than those included in currently available vaccines, a speculation that remains to be examined independently and after a long period of clinical development The steps to make a new vaccine are complicated, costly, time consuming and fraught with many challenges This project has resulted in vaccine candidates that can move into human trials—a key step of the developmental sequence for a rotavirus vaccine that will take several years to accomplish To be compliant with WHO and international standards, we need to consider and develop processes that ensure safe, consistent, robust vaccine production according to GMP practices In addition, safety, immunogenicity and efficacy in human subjects need to be performed according to GCP Finally, this process needs to take into consideration the overall costs and ability to produce this vaccine on a manufacturing scale All of these various activities will be critical for the ultimate success of the project The level of international collaboration and technical support from international partners including CDC and WHO, as we have initiated this rotavirus vaccine project, has enabled us to upgrade many of these aspects including GMP and GLP in the preparation of appropriate cell substrates and techniques, safety and toxicity testing of the oral polio vaccines currently prepared at POLYVAC F138 L.T Le et al / Vaccine 27S (2009) F130–F138 Clearly there are many hurdles ahead in the science, regulatory issues, and clinical testing of these candidates, but the government’s commitment to this internationally collaborative project has helped us think more deeply about how to approach a problem that affects infants throughout the world Acknowledgments We thank Dang Mai Dung, Nguyen Thi Quy, Nguyen Cong Long, and Bui Huy Phuong for technical assistance and Wendi Kuhnert for critical reading of the manuscript Technical support was provided by the Gastroenteritis and Respiratory Viruses Laboratory Branch, Division of Viral Diseases and Biologics Branch, Division of Scientific Resources, the Centers for Disease Control and Prevention, Atlanta, Georgia, USA Conflict of interest None Funding sources Supported by grants form the Ministry of Health and the Ministry of Science and Technology, Vietnam, and the World Health Organisation The findings and conclusions in this report are those of the authors and not necessarily represent the views of U.S CDC References [1] Nguyen 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development of candidate rotavirus vaccines from human strains G1P[8], G1P[4] and G4P[6] isolated in Vietnam These strains were adapted for growth, cloned, and prepared as MS and WS banks in